Method for separating unmodified hemoglobin from cross-linked hemoglobin

ABSTRACT

A method for separating unmodified hemoglobin from cross-linked hemoglobin in a hemoglobin solution. The method involves contacting the hemoglobin solution with a least one dissociating agent to form a dissociation solution wherein unmodified tetrameric hemoglobin is dissociated to form hemoglobin dimers. The hemoglobin dimers are then separated from the dissociation solution, while retaining the cross-linked hemoglobin in the dissociation solution.

RELATED APPLICATIONS

[0001] This application is a Continuation of U.S. Ser. No. 09/460,153,filed on Dec. 13, 1999, which is a Continuation of U.S. Ser. No.09/305,412, filed on May 5, 1999, which is a Continuation of U.S. Ser.No. 08/477,916, filed on Jun. 7, 1995. The entire teachings of the aboveapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] There exists a need for a blood-substitute to treat or preventhypoxia resulting from blood loss (e.g, from acute hemorrhage or duringsurgical operations), resulting from anemia (e.g., pernicious anemia orsickle cell anemia), or resulting from shock (e.g, volume deficiencyshock, anaphylactic shock, septic shock or allergic shock).

[0003] The use of blood and blood fractions as in these capacities as ablood-substitute is fraught with disadvantages. For example, the use ofwhole blood often is accompanied by the risk of transmission ofhepatitis-producing viruses and AIDS-producing viruses which cancomplicate patient recovery or result in patient fatalities.Additionally, the use of whole blood requires blood-typing andcross-matching to avoid immunohematological problems and interdonorincompatibility.

[0004] Hemoglobin, as a blood-substitute, possesses osmotic activity andthe ability to transport and transfer oxygen. However, aqueoushemoglobin exists in equilibrium between the tetrameric (MW 68,000) anddimeric (MW 34,000) forms. Hemoglobin dimers are excreted by the kidneyand result in rapid intravascular elimination of hemoglobin solutionswith such solutions typically having a 2-4 hour plasma half-life.

[0005] Efforts have been directed to overcome the inherent limitationsof hemoglobin solutions by molecularly modifying the hemoglobin.Intramolecularly and intermolecularly cross-linking of hemoglobin hasgenerally reduced renal elimination and increased intravascularretention time.

[0006] However, solutions of cross-linked hemoglobin still typicallycontain a significant fraction of unmodified tetrameric hemoglobin. Thisunmodified tetrameric hemoglobin can convert to dimeric hemoglobin andthen be excreted from the body, thereby reducing the averageintravascular retention time for cross-linked hemoglobinblood-substitutes. Furthermore, current means for separation, such asstandard filtration, do not adequately distinguish between unmodifiedtetrameric hemoglobin and modified tetrameric hemoglobin.

[0007] Thus, in spite of the recent advances in the preparation ofcross-linked hemoglobin blood-substitutes, the need continues to existfor a method to effectively separate unmodified hemoglobin from asolution of an intramolecularly and/or intermolecularly cross-linkedhemoglobin blood-substitute to improve the average intravascularretention time of the blood-substitute and to prevent significant levelsof renal excretion of hemoglobin.

SUMMARY OF THE INVENTION

[0008] The present invention relates to a method for separatingunmodified hemoglobin from cross-linked hemoglobin in a hemoglobinsolution. The method involves contacting the hemoglobin solution with aleast one dissociating agent to form a dissociation solution whereinunmodified tetrameric hemoglobin is dissociated to form hemoglobindimers. The hemoglobin dimers are then separated from the dissociationsolution, while retaining the cross-linked hemoglobin in thedissociation solution.

[0009] The advantages of this invention include providing ablood-substitute with an improved intravascular retention time, areduction or elimination of significant renal elimination of hemoglobinand the side effects associated therewith, a suitable oncotic pressure,and reduced hypertensive effects.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 represents a schematic flow diagram of a method forseparating unmodified hemoglobin from modified hemoglobinblood-substitute according to the present invention.

[0011]FIG. 2 represents the molecular weight distribution of a modifiedhemoglobin polymer, in solution, treated according to the method of thisinvention.

[0012] The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0013] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the spirit and scope of theinvention as defined by the appended claims.

[0014] Hemoglobin (Hb) suitable for Hb solutions of this invention canbe derived from new, old or outdated blood from humans and othervertebrates, such as cattle, pigs, sheep and chickens. In addition,transgenically-produced hemoglobin, such as the transgenically-producedHb described in BIO/TECHNOLOGY, 12: 55-59 (1994), and recombinantlyproduced hemoglobin, such as the recombinantly produced hemoglobindescribed in Nature, 356: 258-260 (1992), are also suitable for Hbsolutions of this invention.

[0015] The blood can be collected from live or freshly slaughtereddonors. Examples of suitable methods for obtaining hemoglobin, derivedfrom red blood cells, are described in U.S. Pat. Nos. 5,084,558 and5,296,465, issued to Rausch et al. The teachings of U.S. Pat. Nos.5,084,558 and 5,296,465 are incorporated herein by reference in theirentirety.

[0016] In a preferred embodiment, hemoglobin is derived from red bloodcells, or recombinant bacteria, as described in U.S. Pat. No. 5,955,581,issued to Rausch et al., the teachings of which are incorporated hereinby reference in their entirety.

[0017] Suitable hemoglobin solutions comprise aqueous solutions ofdissolved Hb wherein the dissolved Hb includes unmodified Hb in additionto modified tetrameric Hb and/or polymerized Hb.

[0018] Unmodified hemoglobin, as defined herein, is hemoglobin in annon-dissociated tetrameric which can dissociate in aqueous solution intoHb dimers, and dissociated Hb dimers. Hb dimers can further dissociatedinto Hb subunits (monomers). Unmodified Hb may be free (not polymerized)within a Hb solution and/or may be intermolecularly cross-linked into apolymer chain within the Hb solution.

[0019] Cross-linked hemoglobin, as defined herein, is which is modifiedand/or polymerized. For unmodified Hb contained in a Hb polymer chain,any dimers which are not intermolecularly cross-linked can bedissociated and separated by the method of invention.

[0020] Modified hemoglobin, as defined herein, is Hb which has beenintramolecularly cross-linked to preclude significant dissociation, inaqueous solution, of Hb tetramers into Hb dimers.

[0021] In polymerized hemoglobin, Hb tetramers are intermolecularlycross-linked to form a Hb polymer chain. A hemoglobin polymer cancontain modified hemoglobin, unmodified hemoglobin, or a combinationthereof. In this method, Hb dimers can be dissociated from unmodified Hbtetramers within a polymer chain, in aqueous solution, if the Hb dimeris not intermolecularly bound to other Hb tetramers.

[0022] In the method of the present invention, at least one dissociationagent is contacted with hemoglobin in an aqueous solution to form adissociation solution. Suitable dissociation agents are water-solubleagents at a concentration within an aqueous solution which, when exposedto unmodified hemoglobin tetramers, result in breaking at least aportion of the hydrogen bonds between Hb dimers in the unmodified Hbtetramers to dissociate the unmodified Hb tetramers into independentα₁β₁ and/or α₂β₂ Hb dimers. The Hb dimers may also further dissociate toform Hb subunits (α₁, α₂, β₁ and β₂).

[0023] A dissociation solution typically contains a concentration ofdissolved dissociation agent having a normality of about 1 gm-equivalentof dissociation agent per liter of dissociation solution, or more.Preferably, a the concentration of dissociation agent within adissociation solution is greater than about 1.4 N.

[0024] Dissociation agents must be ionic or strongly polar when in anaqueous solution. Examples of suitable dissociation agents include,water soluble inorganic salts (e.g., salts of sodium, calcium, magnesiumand zinc), water soluble organic salts (e.g., triethylamine chloride),and water soluble organic amines (e.g., guanidine). Preferably, adissociation agent is an inorganic salt, or salts, containing at leastone multivalent metal cation such as Ca⁺², Mg⁺² or Zn⁺².

[0025] Water soluble, as defined herein, means that the material issufficiently soluble in water at room temperature to form a solutionwith a concentration sufficient, when contacted with Hb, to result inbreaking at least a portion of the hydrogen bonds between Hb dimers inunmodified Hb tetramers.

[0026] The dissociation agent can be dissolved in an aqueous solutionprior to being contacted with the Hb solution or conversely, thedissociation agent can be a solid, in powder or particulate form, whencontacted with an aqueous Hb solution wherein the dissociation agentwill then dissolve.

[0027] Either the dissociation agent or the Hb solution can be added tothe other, or they can be added together. The dissociation agent and theHb solution can be contacted by batch feed or continuous feed.

[0028] In a preferred embodiment, the dissociation agent is mixed withthe Hb solution by a suitable mixing means. More preferably, the mixingmeans in a low shear mixing device as described in U.S. Pat. No.5,955,581, issued to Rausch et al., the teachings of which areincorporated herein by reference in their entirety.

[0029] Examples of suitable hemoglobin solutions include hemoglobinsolutions which have a stabilized 2,3-diphosphoglycerate level, asdescribed in U.S. Pat. No. 3,864,478, issued to Bonhard; cross-linkedhemoglobin, as described in U.S. Pat. No. 3,925,344, issued to Mazur, orin U.S. Pat. Nos. 4,001,200, 4,001,401 and 4,053,590, issued to Bonsenet al., or in U.S. Pat. No. 4,061,736, issued to Morris et al., or inU.S. Pat. No. 4,473,496, issued to Scannon; stroma-free hemoglobin, asdescribed in U.S. Pat. No. 3,991,181, issued to Doczi, or in U.S. Pat.No. 4,401,652, issued to Simmonds et al. or in U.S. Pat. No. 4,526,715,issued to Kothe et al.; hemoglobin coupled with a polysaccharide, asdescribed in U.S. Pat. No. 4,064,118, issued to Wong; hemoglobincondensed with pyridoxal phosphate, as described in U.S. Pat. No.4,136,093, issued to Bonhard et al; dialdehyde-coupled hemoglobin, asdescribed in U.S. Pat. No. 4,336,248, issued to Bonhard et al.;hemoglobin covalently bound with inulin, as described in U.S. Pat. No.4,377,512, issued to Ajisaka et al; hemoglobin or a hemoglobinderivative which is coupled with a polyalkylene glycol or a polyalkyleneoxide, as described in U.S. Pat. No. 4,412,989, issued to Iwashita etal., or U.S. Pat. No. 4,670,417, issued to Iwasaki et al., or U.S. Pat.No. 5,234,903, issued to Nho et al.; pyrogen- and stroma-free hemoglobinsolution, as described in U.S. Pat. No. 4,439,357, issued to Bonhard etal.; stroma-free, non-heme protein-free hemoglobin, as described in U.S.Pat. No. 4,473,494, issued to Tye; modified cross-linked stroma-freehemoglobin, as described in U.S. Pat. No. 4,529,719, issued to Tye;stroma-free, cross-linked hemoglobin, as described in U.S. Pat. No.4,584,130, issued to Bucci et al.; α-cross-linked hemoglobin, asdescribed in U.S. Pat. No. 4,598,064 and Pat. No. Re. 34,271, issued toWalder et al.; stable aldehyde polymerized hemoglobin, as described inU.S. Pat. No. 4,857,636, issued to Hsia; hemoglobin covalently linked tosulfated glycosaminoglycans, as described in U.S. Pat. No. 4,920,194,issued to Feller et al.; modified hemoglobin reacted with a highmolecular weight polymer having reactive aldehyde constituents, asdescribed in U.S. Pat. No. 4,900,780, issued to Cerny; hemoglobincross-linked in the presence of sodium tripolyphosphate, as described inU.S. Pat. No. 5,128,452, issued to Hai et al.; stable, polyaldehydepolymerized hemoglobin, as described in U.S. Pat. No. 5,189,146, issuedto Hsia; and β-cross-linked hemoglobin, as described in U.S. Pat. No.5,250,665, issued to Kluger et al. The teachings of the patents notedabove are hereby incorporated by reference in their entirety.

[0030] Other examples of suitable Hb solutions are described, forexample, in U.S. Pat. No. 5,296,465, issued to Rausch et al and U.S.Pat. No. 5,955,581, issued to Rausch et al. The teachings of U.S. Pat.Nos. 5,296,465 and 5,955,581, issued to Rausch et al, are incorporatedherein by reference in their entirety.

[0031] In a preferred embodiment, hemoglobin used in the method ofinvention is in the form of a polymerized hemoglobin blood-substitute.Examples of suitable polymerized hemoglobin blood-substitutes aredescribed in U.S. Pat. Nos. 5,084,558 and 5,217,648, issued to Rausch etal., U.S. Pat. No. 5,955,581, issued to Rausch et al., and also U.S.Pat. No. 5,895,810, issued to William R. Light et al., the teachings ofwhich are incorporated herein by reference.

[0032] The composition of Hb blood-substitutes preferred for use in themethod of invention are sterile aqueous solutions having less than 0.5endotoxin units/ml, a methemoglobin content that will not result in asignificant reduction in oxygen transport/transfer capacity, a totalhemoglobin concentration between about 1 to about 25 g Hb/dl, aphysiologic pH and a chloride ion concentration of less than 35 meq/l.

[0033] The term “endotoxin” refers to the cell-bound lipopolysaccharidesproduced as a part of the outer layer of bacterial cell walls, whichunder many conditions are toxic. An endotoxin unit (EU) has beendefined, by the United States Pharmacopeial Convention of 1983, page3013, as the activity contained in 0.1 nanograms of U.S. referencestandard lot EC-5. One vial of EC-5 contains 10,000 EU.

[0034] Conditions within the dissociation solution, such as pH andtemperature, are within ranges which will not significantly reduce theability of the Hb to transport and release oxygen, such as would occurfrom denaturing the Hb. Such suitable conditions are as classicallyknown in the art.

[0035] The pH of the dissociation solution must be low enough for Hbtetramer dissociation to occur and high enough to preclude significantacid-induced denaturing of the Hb. Typically, pH is maintained betweenabout 4.5 and about 9.5. Preferably, the pH of the dissociation solutionis acidic.

[0036] In another embodiment, the dissociation solution also contains abuffer to maintain the dissociation solution within a suitable pH range,typically about 4.5 to about 9.5. The buffer can consist of one or morechemical compound.

[0037] A preferred buffer comprises2,2-bis(hydroxy-methyl)-2,2′,2″-nitrilotriethanol (Bis-Tris) with a pHbetween 5.5 and 8.0.

[0038] The dissociation solution can be buffered by adding a solid(powder or particulate) buffer or an aqueous buffer solution to the Hbsolution. Further, the buffer can be added to an aqueous solution of thedissociation agent prior to being contacted with the Hb solution.

[0039] In yet another embodiment, the dissociation solution furthercontains a stabilizing agent in an amount suitable to minimize theformation of methemoglobin by auto-oxidation. An example of a suitableamount of a stabilizing agent is a 0.1 mM solution ofethylenediaminetetraacetic acid (EDTA).

[0040] Hemoglobin solutions used in this method are typically maintainedunder conditions sufficient to minimize microbial growth, or bioburden,such as maintaining temperature at less than about 20° C. and above 0°C. Preferably, temperature is maintained at a temperature of about 15°C. or less. More preferably, the temperature is maintained at about 10°C. to about 12° C.

[0041] The dissociation solution is then filtered to purify the Hbsolution by separating dissociated Hb dimer from modified Hb and/orpolymerized Hb. Suitable filters include ultrafilters which will pass inthe filtrate components having a molecular weight cutout between about40,000 Daltons and 100,000 Daltons. During filtration, components of theHb solution, which are smaller in diameter than modified tetrameric Hb,or which are fluids or dissolved, pass through the filter with thefiltrate. However, the modified Hb tetramers and the polymerized Hbgenerally remains in the retentate.

[0042] A 50,000 Dalton ultrafilter is preferred as it will allowseparation of Hb dimers from the Hb solution without a significant lossof yield of modified Hb tetramers or polymerized Hb.

[0043] In one embodiment, the dissociation solution is only filteredonce. Alternately, the dissociation solution can purified by one thenone filter in series, wherein the retentate of from a previous filter isfurther purified by a subsequent filter.

[0044] Preferably, the Hb retentate is recirculated continuously throughone or more filters, as shown in FIG. 1, thereby continuing to removedimer as unmodified Hb continues to dissociate in the dissociationsolution over time.

[0045] In another embodiment, water or an aqueous solution ofelectrolytes, or preferably of dissociation agent, is added to thedissociation solution before and/or during filtration to at leastparticularly make up for the fluid volume lost as filtrate duringfiltration. The water or aqueous solution can be added batchwise orcontinuously at a rate equal to the rate of filtrate volume loss throughthe filter.

[0046] Water as used in the method of invention may be distilled water,deionized water, water-for-injection (WFI) and/or low pyrogen water(LPW). WFI, which is preferred, is deionized, distilled water that meetsU.S. Pharmacological Specifications for water-for-injection. WFI isfurther described in Pharmaceutical Engineering, 11, 15-23 (1991). LPW,which is more preferred, is deionized water containing less than 0.002EU/ml.

[0047] Typically, about 99%, or more, of the unmodified Hb has beenseparated from the modified Hb and polymeric Hb in the Hb retentate,when the volume of filtrate removed from the Hb solution equals about500% of the volume of the Hb solution prior to adding the dissociationagent.

[0048] When using the Hb solution as a blood-substitute, the hemoglobinin the Hb retentate is then washed and equilibrated by diafiltrationwith a physiologic buffer to ensure the physiological acceptability ofthe blood-substitute. Suitable physiologic buffers include buffers thathave physiologically acceptable levels of electrolytes (e.g, NaCl, KCland CaCl₂) in WFI. Preferably, the buffer is depyrogenated, such as byfiltration with a 10,000 Dalton ultrafilter, and deoxygenated.

[0049] A buffer solution can further include a dissolved, non-toxicreducing agent, such as N-acetyl-L-cysteine, cysteine, sodium dithioniteor ascorbate, to chemically scavenge oxygen in the blood-substitute toreduce methemoglobin formation. For Hb blood-substitutes, amethemoglobin content of about 25% or more will typically result in asignificant reduction in oxygen delivery capacity. It is preferred thatmethemoglobin content be less than about 15%. In an even more preferred,the methemoglobin content in a Hb blood-substitute be less than or equalto about 10%.

[0050] Oxygenation of Hb, similar to dissociation buffers, will alsodissociate unmodified hemoglobin into Hb dimers, as shown in ExampleIII. However, oxygenation of Hb also promotes methemoglobin formation.

[0051] It is understood that the physiologic buffer and the reducingagent can be added separately, or jointly, to the Hb retentate in abatch or continuous feed mode. In a preferred embodiment, the Hbretentate is washed by diafiltration against a physiologic buffer untilthe Hb solution is physiologically acceptable to humans and/or othervertebrates.

[0052] Typically, diafiltration continues until the volume of fluid lostthrough diafiltration across the diafilter is about five times, or more,of the initial volume of the Hb retentate before washing. In a morepreferred embodiment diafiltration is continued until about 10 volumesof fluid have been exchanged.

[0053] Further description of the use of this method, to removeunmodified Hb from polymeric Hb solutions, is provided in Examples I andII.

[0054] In this method, portions of the components for the process for apreparing a stable polymerized hemoglobin blood-substitute aresufficiently sanitized to produce a sterile product. Sterile is asdefined in the art, specifically, that the solution meets United StatesPharmacopeia requirements for sterility provided in USP XXII, Section71, pages 1483-1488. Further, portions of components that are exposed tothe process stream, are usually fabricated or clad with a material thatwill not react with or contaminate the process stream. Such materialscan include stainless steel and other steel alloys, such as Inconel.

[0055] The pump used in this method can be a peristaltic-type,diaphragm-type, gear-type, piston-type or rotary-lobe type pump.Diaphragm-type pumps are available from Branne Lubbem Inc., BuffaloGrove, Ill. Suitable rotary-lobe pumps include the Albin SLP 110 P51 B1sanitary lobe-rotary pump from Albin Pump Inc., Atlanta, Ga. Rotary-lobepumps can also be obtained from Waukesha Pumps, Waukesha, Wis.

[0056] One embodiment of a system 10, suitable for practicing the methodof invention for separating unmodified hemoglobin from a Hb solutioncontained modified Hb tetramer and/or polymeric Hb, is illustrated inFIG. 1. System 10 includes tank 12, pump 14, purification filter 16 anddiafilter 18. Pump 14 takes a suction on tank 12 and recirculates Hbsolution through purification filter 16 and/or diafilter 18. It isunderstood that purification filter 16 and diafilter 18 can be operatedin parallel or in series arrangements. Tank 12 contains Hb solutionwhich can be formed within tank 12 or which can be formed prior to beingtransferred into tank 12.

[0057] An amount of an Hb dissociation agent, suitable to dissociate Hbtetrameric molecules into Hb dimers, is then contacted with the Hbsolution in system 10 to form a dissociation solution. The dissociationagent is typically introduced into tank 12, from dissociation agentsupply 20. However, it is understood that the dissociation agent can beadded at other locations in system 10. It is also understood that thedissociation agent can be added to the Hb solution in a batch orcontinuous feed mode.

[0058] The dissociation agent and the Hb solution in the dissociationsolution are then mixed by a low shear mixing, specifically static mixer22. The dissociation agent and the Hb solution are mixed byrecirculating the dissociation solution from tank 12, by pump 14 throughan orifice, not shown, and static mixer 22.

[0059] Static mixer 22 is typically located downstream of pump 14 andupstream of purification filter 16, however, static mixer 22 alternatelycould be located at other points in system 10.

[0060] The unmodified hemoglobin in the dissociation solution thencommences to dissociate from unmodified Hb tetramers into Hb dimers. TheHb dimers each have a molecular weight of about 32,000 Daltons.

[0061] The dissociation solution is then recirculated throughpurification filter 16 to remove Hb dimers in the filtrate, and retainmodified Hb and polymeric Hb in the Hb retentate. During filtration,components of the dissociation solution, which are smaller in diameterthan stabilized tetrameric Hb, or which are fluids, pass throughpurification filter 16 with the filtrate. Examples of suitablepurification filters include ultrafilters with a molecular weight cutoutbetween about 40,000 Daltons and about 100,000 Daltons.

[0062] In a continuous feed mode, a liquid or dissolved dissociationagent is added continuously, as makeup, at a rate equal to the rate offiltrate loss through purification filter 16. In another embodiment, thevolume of filtrate discharged through purification filter 16 isregulated by filtrate pump 24.

[0063] Separation of unmodified Hb from the Hb retentate is typicallycomplete when the volume of filtrate drained from purification filter 16equals about 500% of the volume of Hb solution contained in tank 22prior to adding dissociation agent to system 10.

[0064] In another embodiment, the Hb retentate is then washed andequilibrated by diafiltration with a physiologic buffer to make the Hbretentate physiologically acceptable as a blood-substitute. Aphysiologic buffer is introduced into tank 12, from physiologic buffersupply 26. However, it is understood that the physiologic buffer can beadded at any location in system 10. It is also understood that thephysiologic buffer can be added to the Hb retentate in a batch orcontinuous feed mode.

[0065] A preferred physiologic buffer includes 27 mM sodium lactate, 12mM N-acetyl-L-cysteine, 115 mM NaCl and 1.36 mM CaCl₂ in WFI (pH 8).

[0066] The Hb retentate is then diafiltered by recirculating the Hbretentate and physiological buffer from tank 12, by pump 14 throughstatic mixer 22 and diafilter 18. Diafilter 18 is located downstream ofstatic mixer 22 and upstream of tank 12. Diafiltration continues untilthe blood-substitute is physiologically acceptable. Typically, theblood-substitute is physiologically acceptable when the volume of fluidlost through diafiltration across diafilter 18 is at least five timesthe initial volume of the Hb retentate in system 10.

[0067] During Hb dissociation and Hb retentate diafiltration, the Hbtemperature is maintained at approximately 8° C. to 12° C. in tank 14.An example of an acceptable means for controlling the Hb temperature isby cooling the outside of tank 14 through use of an ethylene glycoljacketed cooling system, not shown.

[0068] The invention will be further illustrated by the followingnon-limiting examples.

EXAMPLE I Diafiltration of Deoxygenated Hb Solution Containing a HigherConcentration Dissociation Buffer

[0069] A polymerized Hb solution was formed according to the methoddescribed in Example 1 of U.S. Pat. No. 5,084,558, issued to Rausch etal. This Hb solution was analyzed by gel permeation chromatography (GPC)and found to comprise about 45% Hb dimers, about 15% unmodified Hbtetramers, and about 40% polymerized Hb molecules which were larger thanunmodified tetramers. One liter of a dissociation buffer containing 1.5M MgCl₂, 0.1 M Bis-Tris and 0.2 mM EDTA (pH 6.5) was added to one literof the Hb solution. This mixture was then recirculated through a 100 kDpolysulfone ultrafilter (Millipore Catalog No. PTHK 000C5) toconcentrate the mixture to a volume of one liter. The concentratedmixture was subsequently diafiltered with 11 volumes of a dissociationbuffer comprising 0.7 M MgCl₂, 0.05 M Bis-Tris and 0.1 mM EDTA (pH 6.5).The filtered Hb solution was then washed and equilibrated with adeoxygenated buffer containing 27 mM sodium lactate, 12 mM N-acetylcysteine, 115 mM NaCl, 4 mM KCl, and 1.36 mM CaCl₂ in WFI. The molecularweight distribution of the resulting Hb solution was then analyzed byGPC.

[0070] The results of these analyses are shown in FIG. 2. The Hbsolution was then found to have a final composition of about 5% Hbdimers, about 10% Hb tetramers and about 85% polymerized Hb moleculeswhich were larger than tetramers.

EXAMPLE II Diafiltration of Deoxygenated Hb Solution Containing a LowerConcentration Dissociation Buffer

[0071] One hundred seventy milliliters (ml) of a dissociation buffercontaining 0.75 M MgCl₂, 0.05 M Bis-Tris and 0.1 mM EDTA (pH 7.5) wasadded to 15 ml of the initial polymerized Hb solution of Example I andthen 15 ml of a two-fold concentrate of the dissociation buffer wasadded. The Hb solution was then recirculated through a Chemineer,Inc./Kenics static mixer and then diafiltered by a 100 kD ultrafilter(Amicon YM 100, Catalog No. 14451) to obtain 200 ml of Hb solution.

[0072] The Hb solution was then diafiltered with 3 volume exchanges ofthe dissociation buffer and lastly washed and equilibrated with adeoxygenated buffer containing 27 mM sodium lactate, 12 mM N-acetylcysteine, 115 mM NaCl, 4 mM KCl, and 1.36 mM CaCl₂ in WFI. The molecularweight distribution of the resulting Hb solution was then analyzed byGPC.

[0073] The results of these analyses are shown in FIG. 2. The Hbsolution was then found to have a final composition of about 15% Hbdimers, about 15% Hb tetramers and about 70% polymerized Hb moleculeswhich were larger than tetramers. Thus, the method of invention waseffective in reducing the content of unmodified hemoglobin in thepolymerized Hb solutions.

EXAMPLE III Diafiltration of Oxygenated and Deoxygenated Hb SolutionsWithout a Dissociation Buffer

[0074] A polymerized Hb solution was formed according to the methoddescribed in Example 1 of U.S. Pat. No. 5,955,581, issued to Rausch etal. This Hb solution was analyzed by GPC and found to comprise about3.5% Hb dimers, 31% unmodified Hb tetramers and about 65.5% polymerizedHb molecules which were larger than unmodified tetramers.

[0075] Two liters of the Hb solution were oxygenated through anoxygenation cartridge with a gaseous mixture, comprising 98% oxygen and2% carbon dioxide, until 95% oxygenated Hb valves were obtained by aco-oximeter (Co-Oximeter Model #482; Instrumentation Laboratory,Lexington, Mass.).

[0076] The oxygenated Hb solution was then diafiltered with 7 volumes ofan oxygenated buffer solution containing 27 mM solution lactate, 12 mMN-acetyl-L-cysteine, 115 mM NaCl, 4 mM KCl and 1.4 mM CaCl₂ in WFIagainst a 100 kD ultrafilter. Throughout this process, the Hb solutionwas not contacted with any dissociation buffer.

[0077] The molecular weight distribution of the resulting Hb solutionwas then analyzed by GPC. The molecular weight distribution was found tobe 0.5% dimer and 2.7% unmodified tetramer and about 96.8% polymerizedHb molecules which were larger than unmodified tetramers.

[0078] This entire procedure was then repeated on another sample of thesame polymerized Hb solution, with the exception that the Hb solutionwas not oxygenated prior to diafiltration. The molecular weightdistribution of the resulting Hb solution was found by GPC to be 2.2%dimer, 2.5% unmodified tetramer and about 95.3% polymerized Hb moleculeswhich were larger than unmodified tetramers.

[0079] The results of these procedures show that oxygenation ofhemoglobin likewise promotes the dissociation of unmodified Hb tetramerto Hb dimers. However, oxygenation also promotes the formation of theundesirable methemoglobin.

EXAMPLE IV Molecular Weight Analysis

[0080] Molecular weight was determined by conducting gel permeationchromatography (GPC) on the hemoglobin solutions under dissociatingconditions. This method of analysis results in the separation ofhemoglobin polymers on the basis of size, with larger molecules elutingfaster than smaller molecules. By comparison to protein molecular weightstandards, it is possible for correlate elution time with the molecularweights of the hemoglobin products.

[0081] In this analysis, a representative sample of the hemoglobinproduct was analyzed for molecular weight distribution. The hemoglobinproduct was diluted to 4 mg/ml within a mobile phase of 50 mM Bis-Tris(pH 6.5), 750 mM MgCl₂, and 0.1 mM EDTA. The diluted sample was injectedonto a HPLC TosoHaas G3000SW column. Flow rate is 0.5 ml/min. andultraviolet detection was set at @280 nm.

EQUIVALENTS

[0082] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. These and allother such equivalents are intended to be encompassed by the followingclaims.

What is claimed is:
 1. A method for separating unmodified hemoglobinfrom cross-linked hemoglobin in a hemoglobin solution, comprising thesteps of: a) contacting the hemoglobin solution with a least onedissociating agent to form a dissociation solution wherein unmodifiedtetrameric hemoglobin is dissociated to form hemoglobin dimers; and b)separating the hemoglobin dimers from the dissociation solution, whileretaining the cross-linked hemoglobin in said dissociation solution. 2.A method of claim 1 wherein at least a portion of the hemoglobin in thehemoglobin solution is polymerized.
 3. A method of claim 1 wherein thedissociation agent is a water soluble inorganic salt.
 4. A method ofclaim 3 wherein the inorganic salt is a sodium salt.
 5. A method ofclaim 3 wherein the inorganic salt includes a multivalent cation.
 6. Amethod of claim 5 wherein the multivalent cation is selected from thegroup consisting of Ca⁺², Mg⁺², Zn⁺² and combinations thereof.
 7. Amethod of claim 1 wherein the dissociation agent is a water solubleorganic salt.
 8. A method of claim 7 wherein the organic salt is a saltof an amine.
 9. A method of claim 1 wherein the dissociation agent is awater soluble organic amine.
 10. A method of claim 9 wherein the amineis guanidine.
 11. A method of claim 1 further comprising the step ofmixing the dissociation agent and the hemoglobin solution under lowshear conditions.
 12. A method of claim 1 further comprising the step ofcontacting the hemoglobin solution with a buffer.
 13. A method of claim12 wherein the Hb solution is buffered with2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol.
 14. A method of claim1 further comprising the step of contacting the Hb solution with astabilizing agent.
 15. A method of claim 14 wherein the stabilizingagent is ethylenediaminetetraacetic acid.
 16. A method of claim 1wherein the unmodified hemoglobin is separated from the dissociationsolution by filtering the dissociation solution.
 17. A method of claim 1further comprising the step of washing the cross-linked hemoglobin witha physiologically acceptable buffer, after separating the unmodifiedhemoglobin from the dissociation solution, to produce a physiologicallyacceptable hemoglobin blood-substitute.
 18. A method for separatingunmodified hemoglobin from cross-linked in a hemoglobin solution toproduce a physiologically acceptable blood-substitute, comprising thesteps of: a) contacting the hemoglobin solution with a dissociatingagent to form a dissociation solution, wherein unmodified tetramerichemoglobin dissociated to form hemoglobin dimers; b) filtering thehemoglobin dimers from the dissociation solution while retainingcross-linked hemoglobin in the retentate; and c) washing thecross-linked hemoglobin with a physiologic buffer to produce aphysiologically acceptable blood-substitute.
 19. A method of claim 19wherein the buffer contains sodium lactate and N-acetyl-L-cysteine. 20.A method for separating unmodified hemoglobin from cross-linkedhemoglobin in a hemoglobin solution, comprising the steps of: a) mixingthe hemoglobin solution with a dissociation agent, a buffer and astabilizing agent to form a dissociation solution, wherein mixing iswith low shear, and wherein the unmodified hemoglobin dissociates toform hemoglobin dimers within the dissociation solution; b) filteringthe dissociation solution to separate the hemoglobin dimers from thecross-linked hemoglobin; and e) washing the cross-linked hemoglobinsolution with a deoxygenated solution containing sodium lactate,N-acetyl-L-cysteine and physiologic electrolytes.